1. Field of the Invention
This invention is in the field of assays useful for the screening of chemical compounds able to serve as immunosuppressive agents.
The present invention arises from the discovery of a method for the identification of agents that selectively induce apoptosis in activated T lymphocytes. Compounds of this nature are useful as therapeutically effective immunosuppressive agents. In particular, the invention relates to the use of primary T cell cultures to identify compounds that directly or indirectly activate the caspase cascade.
Also taught are methods for using the immunosuppressive agents and pharmaceutical compositions for their use.
2. Related Art
Immunopathological Diseases: The immune system is a remarkably evolved defense system in vertebrates for protection against pathogenic microorganisms. The same immune system can also lead to various pathological conditions. For example, the immune system can cause rejection of grafts during transplantation (Rosenberg, A. and S. Singer, Annu. Rev. Immunol. 10:333 (1992)). Also, graft versus host disease (GvHD) develops when a graft containing immunocompetent T cells recognize and react with the recipient""s cells (Woo, S.-B., et al., Crit. Rev. Oral. Biol. Med. 8:201 (1997)). Mechanisms of self-tolerance normally protect an individual from self-reactive T lymphocytes. However, should these mechanisms fail, an inappropriate immune response occurs leading to what is known as autoimmunity. Rheumatoid arthritis is a well-known example of autoimmunity. In this degenerative condition, auto-reactive T cells destroy the tissue around the joints causing inflammation and tissue destruction.
Transplantation or Graft Rejection: A problem arises during kidney, cardiac, lung or liver transplants and skin grafts when the host immune system recognizes the transplant graft as foreign tissue and develops immune reactivity that ends in rejection of the transplanted/grafted tissue. Several attempts are being made to induce immunological tolerance across the major histocompatibility complex (MHC) barriers. This is generally achieved by three mechanisms:
1) clonal deletion of the activated antigen/MHC reactive lymphocytes:
2) clonal anergy and suppression on the other hand by antibody mediated blockade of the gene expression; or
3) Suppression of one subset of the T cells (Th1) and expansion of the other (Th2) in situations of cardiac allografts studies were also partly successful (Bach. F., et al., Nat. Med. 3:196-204 (1997); Sayegh, M H., et al., J. Exp. Med. 181:1869-1874 (1995).
Graft-Versus-Host Disease (GvHD): GvHD is the most important complication of bone marrow transplantation (BMT) (Ferrara, J. and H. Deeg, N. Engl. J. Med. 324:667 (1991)). When competent T cells are transferred from a donor to a recipient who is incapable of rejecting them, the grafted cells survive, start recognizing the host antigens and develop immune reactivity towards them. Instead of the normal transplantation reaction of host versus graft, the reverse is seen in this case. Research indicates that increased donor T cell and monocyte/macrophage expansion and inflammatory cytokines are responsible for this syndrome (Via, C., et al, J. Immunol. 157:5387 (1996): Krenger, W., et al, Transplantation 64:553 (1997); Hattori, K., et al., Blood 91:4051 (1998); Mori, T., et al., Blood 92:101 (1998)).
Autoimmune Diseases: Among this group of diseases is rheumatoid arthritis which is a chronic inflammatory disease of the joints, characterized by infiltration of T lymphocytes into the synovial fluid and eventual destruction of the cartilage and bones in the affected joints. Several studies have suggested that the infiltrating T lymphocytes are activated and cause neighboring tissue destruction. Other autoimmune diseases due to autoreactive T lymphocytes include multiple sclerosis, insulin-dependent diabetes mellitus, lupus, and muscular dystrophy (Liblau, R., et al., Immunol. Today 16:34 (1995)).
Immunosuppressive Agents: Current immunosuppressive treatments result in generalized immunosuppression and leave the patient prone to various infections. These therapies are also aimed at slowing down the proliferation of activated T cells and thereby due to lack of specificity, effect the growth of all normal dividing cells and result in side effects and toxicity. The primary methods of treatment for immunopathological disorders such as graft or transplantation rejection. GvHD and rheumatoid arthritis are corticosteroid and immunosuppressive agents. Current immunosuppressive drugs like cyclosporin A (CsA) and FK506 work by blocking a calcium dependent protein phosphatase calcineurin (Cn), but they often have unwanted side effects such as cancer, kidney failure, and diabetes. Progress is being made in enhancing the effectiveness of each of these agents.
Despite reduced side effects from immunosuppression, certain tissue transplantations still result in morbidity and mortality. Because of the frequent occurrence of corticosteroid related side effects in transplant patients, alternative therapeutic agents are desirable for these and other related disorders. One such therapeutic agent is methotrexate (MTX), a folate antagonist first developed for malignancies (Farber, S., et al., Advances in Cancer Res. 2-73 (1956)) and subsequently used as an anti-inflammatory and/or immunosuppressive drug. MTX is now the most commonly used treatment for rheumatoid arthritis (Weinblatt, M., et al., N. Engl. J. Med. 312:818-822(1985); Williams, B., et al., Arthritis Rheum. 28:721-730 (1985)).
Apoptosis: A normal checkpoint in the life of cells in multicellular organisms is the process of apoptosis (see, e.g., Evan and Littlewood, Science 281:1317-1322 (1998)). Apoptosis is the highly conserved mechanism by which cells commit suicide. Characteristics of the process include an execution phase that includes loss of cell volume, plasma membrane blebbing and chromatin condensation, followed by packing of the cellular contents into membrane-enclosed vesicles called apoptotic bodies that are rapidly phagocytosed by neighboring cells. Apoptosis differs from necrosis, which is cell death resulting from physical injury.
Since autoimmune diseases and certain degenerative diseases also involve the proliferation of abnormal cells, therapeutic treatment for these diseases could also involve the enhancement of the apoptotic process through the administration of appropriate drugs.
It is pertinent, therefore, to inquire into the mechanism of apoptosis in order to develop a method for the identification of compounds for the treatment of autoimmune diseases. It has been found that a group of proteases are a key element in apoptosis (see e.g. Thornberry, Chemistry and Biology 5:R97-R103 (1998); Thornberry, British Med. Bull. 53:478-490 (1996)). Genetic studies in the nematode Caenorhabditis elegans revealed that apoptotic cell death involves at least 14 genes, two of which are the pro-apoptotic (death-promoting) ced (for cell death abnormal) genes, ced-3 and ced-4. CED-3 is homologous to interleukin 1xcex2-converting enzyme (ICE), a cysteine protease, which is now called caspase-1. When these data were ultimately applied to mammals, and upon further extensive investigation, it was found that the mammalian apoptosis system appears to involve a cascade of caspases, or a system that behaves like a cascade of caspases. At present, the caspase family of cysteine proteases comprises 10 different members, and more may be discovered in the future. All known caspases are synthesized as zymogens that require cleavage at an aspartyl residue prior to forming the active enzyme. Thus, caspases are capable of activating other caspases, in the manner of an amplifying cascade.
The caspase cascade can be involved in disease processes in two major aspects. Excessive activity of the caspase cascade can lead to excessive apoptosis and organ failure. Among the diseases that could result from this excessive activity are myocardial infarction, congestive heart failure, viral infections, rheumatoid arthritis and others. Inhibitors of the caspase cascade could thus be candidates for therapeutic intervention in such diseases.
Caspase Cascade Activators: Although the development of enzyme inhibitors as therapeutic agents is a well-understood art (see Muscate and Kenyon, Burger""s Medicinal Chemistry 1:733-782, 5th Ed. (1995)) this is not the case in the development of enzyme activators. The theoretical basis for the development of enzyme activators is still in its infancy. In the case of the apoptosis process, control points are known to exist that represent points for intervention leading to activation. These control points include the CED-9-BCL-like and CED-3-ICE-like gene family products, which are intrinsic proteins regulating the decision of a cell to survive or die and executing part of the cell death process itself, respectively (see Schmitt, et al. Biochem. Cell. Biol. 75:301-314 (1997)). BCL-like proteins include BCL-xL and BAX-xcex1, which appear to function upstream of caspase activation. BCL-xL appears to prevent activation of the apoptotic protease cascade, whereas BAX-xcex1 accelerates activation of the apoptotic protease cascade. Although the exact mechanisms are not fully understood, it is clear that the possibility exists for the activation of the caspase cascade. Because insufficient activity of the caspase cascade and consequent apoptotic events appear to be implicated in various types of immunopathological, degenerative and autoimmune diseases, the development of caspase cascade activators is a highly desirable goal in the development of potentially therapeutically effective immunosuppressive agents.
Assays for Detecting Caspase Cascade Activating Drugs: In order to find drugs that either inhibit or stimulate the caspase cascade, it is necessary to develop high-throughput caspase activation (HTCA) assays. These HTCA assays must be able to monitor activation or inhibition of the caspase cascade inside living cells. Ideally, HTCA assays should be versatile enough to measure the caspase cascade activity inside any living cell, no matter what its origin might be: cancer cells, tumor cells, immune cells, brain cells, cells of the endocrine system, cells or cell lines from different organ systems, biopsy samples, etc. Furthermore, such HTCA assays should be able to measure within living cells the activation or inhibition of any of the caspase enzymes or any other enzymes that are involved in the caspase cascade. Developing such versatile HTCA assays represents a substantial advance in the field of drug screening.
Most HTCA assays do not permit intracellular screening for compounds that can either activate or inhibit the caspase cascade. These assays are typically cell-free, high-throughput screening assays to measure the activity of individually isolated caspase enzymes, or assays that can measure the activity of caspases in dead cells which have been permeabilized by osmotic shock (see Los, et al., Blood, 90:3118-3129 (1997)). But these enzyme assays cannot predict the effect of a compound on the caspase cascade in living cells for the following reasons:
1) Cell free assays, or assays using dead, permeabilized cells, cannot predict the ability of compounds to penetrate the cellular membrane. This is crucial because the caspase cascade resides in the interior of the cells. In order to be active, a compound must not only be able to modulate the caspase enzyme or enzymes, but it must also be able to penetrate the intact cell membrane. Cell-free assays or assays using dead cells are therefore unable to determine whether or not a compound will be potentially useful as a drug.
2) Isolated caspases in cell-free assays are highly susceptible to oxidation and to compounds that can cause oxidation of the enzymes. This property of isolated caspases makes cell free caspase screening assays highly susceptible to artifacts and has precluded successful use of these assays for high-throughput screening of combinatorial (or other) chemical libraries. Previous mass screening efforts, using cell-free caspase enzyme assays, have led to discovery of numerous inhibitors which oxidize caspases, but no compound that would be useful as a potential drug. Others have reported similar difficulties.
3) Numerous cellular receptors, proteins, cell constituents and cofactorsxe2x80x94many of which are still unknownxe2x80x94can influence the caspase cascade in living cells. Cell-free caspase assays or assays using permeabilized, dead cells do not take into account these cellular receptors and cofactors. Because of this, it is possible that a compound identified in a cell-free or dead-cell caspase assay will not work in living cells. On the other hand, a compound that might inhibit or stimulate the caspase cascade indirectly through one of the cellular receptors or cofactors would be missed entirely in a cell-free or dead-cell caspase assay.
4) It is highly likely that the caspase cascade functions differently in cells derived from different organs. There is growing evidence that the receptors and cofactors that influence the caspase cascade differ among cell types. Using cell-free or dead cell assays, it would be virtually impossible to identify cell-type or organ specific modulators of the caspase cascade.
U.S. Pat. No. 6,077,684 discloses a method of measuring the apoptosis-inducing activity of a substance using cultured, isolated cells having intact membranes. This method involves obtaining a sample of cells from a subject; isolating a single cell suspension from the sample; placing the cells in culture conditions; exposing the cells in culture to the putative apoptosis-inducing substance; incubating the cultured cells; measuring in a serial manner the optical densities of the culture to obtain an optical density curve; and correlating the slope of a line representing an increase over time in optical density, due to cellular membrane distortion and blebbing, with an increase in apoptotic activity.
U.S. Pat. No. 6,342,611 and WO 99/18856 disclose a whole cell assay wherein a fluorogenic or fluorescent reporter compound is used to measure the activity of intracellular caspases or other enzymes involved in apoptosis in living or dead whole cells or tissues. In this process, test substances, which may directly or indirectly induce apoptosis, are brought into contact with cells having intact membranes. If one or more of the substances is capable of inducing apoptosis, then intracellular caspase proteases are generated. The reporter compound serves as a substrate for these proteases and fluoresces after being cleaved. The reporter molecules can also be used to measure baseline caspase activity in cells that are not undergoing induced apoptosis. Hence, apoptosis inducing agents may be discovered by monitoring changes in fluorescence occurring within the cells. This process may be used to find new compounds or new uses for known compounds in reducing, preventing or treating maladies in which apoptotic cell death is either a causative factor or a result.
The present invention is directed to methods for identifying direct and indirect activators of the caspase cascade in T cells, therapeutic methods employing such activators, compositions comprising such activators, and kits comprising such activators.
In particular, the invention provides a method for identifying immunosuppressive compounds by determining the ability of test compounds to selectively activate the caspase cascade in activated, viable T cells to a greater extent than in resting viable T cells. Test compounds which are capable of acting outside of the cell, at the cellular membrane, or within the cell to directly or indirectly induce the caspase cascade may be identified due to the presence of intracellular proteases generated as a result of the caspase cascade. Such proteases cleave a reporter molecule which serves as a substrate of the proteases.
More particularly, the invention relates to a method for identifying an immunosuppressive agent comprising obtaining at least one population of viable cultured active T cells having intact cell membranes from a cell growth medium under conditions conducive to growth; combining a first portion of the at least one population with a predetermined amount of at least one test compound dissolved in a solvent for a predetermined period of time at a predetermined temperature thereby generating a first volume; combining a second portion of the at least one population with an amount of the solvent which was used to dissolve the at least one test compound, for the predetermined period of time at the predetermined temperature thereby generating a second volume; separately adding to each of the first volume and the second volume a reporter compound having at least one measurable property which is responsive to the caspase cascade; measuring the at least one measurable property of the reporter compound in the first volume and thereby measuring the caspase cascade activity of the first volume; measuring the at least one measurable property of the reporter compound in the second volume and thereby measuring the caspase cascade activity of the second volume; calculating a first ratio of caspase cascade activity measured for the first volume to the caspase cascade activity measured for the second volume, wherein when the first ratio is greater than one, the at least one test compound kills T cells and is identified as a potential immunosuppressive agent.
Subsequent to identifying a potential immunosuppressive agent, the invention comprises obtaining at least one population of viable cultured resting T cells having intact cell membranes from a cell growth medium under conditions conducive to growth; combining the resting T cells with the predetermined amount of the identified immunosuppressive agent dissolved in the solvent for the predetermined period of time at the predetermined temperature thereby generating a third volume; adding to the third volume the reporter compound having at least one measurable property which is responsive to the caspase cascade; measuring the at least one measurable property of the reporter compound in the third volume and thereby measuring the caspase cascade activity of the third volume; and, calculating a second ratio of caspase cascade activity measured for the first volume to the caspase cascade activity measured for the third volume, wherein when the second ratio is greater than one, then the identified immunosuppressive agent is further identified as an active-T-cell-selective immunosuppressive agent.
The invention also relates to a method for identifying an immunosuppressive agent comprising obtaining at least one population of viable cultured active T cells having intact cell membranes from a cell growth medium under conditions conducive to growth; combining a first portion of the at least one population with a predetermined amount of at least one test compound dissolved in a solvent for a predetermined period of time at a predetermined temperature thereby generating a first volume; combining a second portion of the at least one population with an amount of the solvent which was used to dissolve the at least one test compound, for the predetermined period of time at the predetermined temperature thereby generating a second volume; separately assessing the cell viability of the first volume and the second volume; and comparing the cell viability of the first volume to the cell viability of the second volume, wherein when the cell viability of the first volume is less than the cell viability of the second volume, the at least one test compound kills T cells and is identified as a potential immunosuppressive agent.
Subsequent to identifying a potential immunosuppressive agent, the invention relates to a method of obtaining at least one population of viable cultured resting T cells having intact cell membranes from a cell growth medium under conditions conducive to growth; combining the resting T cells with the predetermined amount of the identified immunosuppressive agent dissolved in the solvent for the predetermined period of time at the predetermined temperature thereby generating a third volume; assessing the cell viability of the third volume; and comparing the cell viability of the first volume to the cell viability of the second volume, wherein when the cell viability of the first volume is less than the cell viability of the second volume, then the identified immunosuppressive agent is further identified as an active-T-cell-selective immunosuppressive agent. Cell viability may be assessed by observing mitochondrial activity, membrane intactness, or cell number. Mitochondrial activity, membrane intactness, or cell number may be measured by using fluorescence methodology, calorimetric assays, or direct visualization techniques, and by using a reporter compound selected from the group consisting of a fluorogenic compound that produces fluorescence under the influence changes in mitochondrial activity, membrane intactness, or cell number; a chromogenic compound that produces light absorption under the influence of changes in mitochondrial activity, membrane intactness, or cell number; and a chemiluminescent compound that produces light emission under the influence of changes in mitochondrial activity, membrane intactness, or cell number.
The invention also relates to a method for assaying the potency of a test compound to synergise with a known immunosuppressant by functioning as an activator of the caspase cascade, comprising obtaining at least one population of viable cultured active T cells having intact cells by culturing T cells in a cell growth medium under conditions conducive to growth and activating the cells; exposing a first portion of the at least one population to a combination of a predetermined amount of the test compound and a subinducing amount of the known immunosuppressant for a first predetermined period of time, at a first predetermined temperature thereby generating a first volume; exposing a second portion of the at least one population to an amount of solvent which was used to dissolve the test compound and to the subinducing amount of the known immunosuppressant for the first predetermined period of time at the first predetermined temperature thereby generating a second volume; adding a reporter compound to the first volume and to the second volume, the reporter compound having at least one measurable property which is responsive to the caspase cascade; incubating the resulting mixture of the first volume with the reporter compound for a second predetermined time period at a second predetermined temperature; incubating the resulting mixture of the second volume with the reporter compound for the second predetermined time period at the second predetermined temperature; measuring the at least one measurable property of the reporter compound in each of the resulting mixtures and thereby measuring the caspase cascade activity of the first volume and of the second volume; and, calculating the ratio of measured caspase cascade activities of the first volume to the second volume to determine whether the test compound synergises with the known immunosuppressant as an activator of the caspase cascade.
The invention also relates to a method for identifying an immunosuppressive agent by determining the ability of at least one test compound to activate the caspase cascade in active T cells having intact cell membranes, comprising obtaining viable cultured active T cells having an intact cell membrane; obtaining viable cultured resting T cells having an intact cell membrane; separately exposing the active and resting T cells to at least one test compound for a predetermined period of time under predetermined conditions; adding a reporter compound having at least one measurable property which is responsive to the caspase cascade to the active and resting T cells; and measuring the caspase cascade activity in the active T cells by measuring the at least one measurable property; measuring the caspase cascade activity in the resting T cells by measuring the at least one measurable property; wherein when the caspase cascade activity in the active cells is greater than the caspase cascade activity in the resting cells, the at least one test compound selectively kills active T cells and is an immunosuppressive agent.